Download Notochordal Cell Marker Expression in The Adult Human Nucleus

Notochordal Cell Marker Expression in the Adult Human Nucleus Pulposus
1
Ludwinski, FE; 2Gnanalingham, K; 1Richardson, SM; 1Freemont AJ; +1Hoyland JA
2
+1University of Manchester, Manchester, UK
Salford Royal Hospital Trust, Manchester, UK
[email protected]
METHODS:
27 lumbar (aged 25 to 75 years) and 27 cervical NP samples (aged
33-72 years) were obtained with informed consent during discectomy in
accordance with university and ethical committee policy and HTA
legislation. Tissue samples were partially processed for histological
analysis or enzymatically digested with RNA extracted from cells and
reverse transcribed to cDNA. Fixed specimens were then histologically
graded1, whilst QPCR was performed on extracted samples for
notochordal markers (Brachyury (T), Galectin-3 (LSGAL3), CD24,
Chordin (CHRD), FOXA2 and NOGGIN). All data was normalised to
expression of the housekeeping genes MRPL19 and EIF2B1, and
analysed according to the ΔCt method. Mann-Whitney tests were
performed to assess statistical significance and p values equal to <0.05
were deemed significant.
RESULTS:
Expression of all NC markers was identified in both lumbar and
cervical NP cells, although T and FOXA2 expression was not detected
in every sample. In cervical NP samples, increased degeneration did not
significantly alter expression of most NC markers, although a significant
increase in NOGGIN expression was noted (Figure 1). A similar pattern
of expression was noted in lumbar specimens. There were also no
significant age-associated alterations in NC marker expression in lumbar
samples (Figure 2). Interestingly, some differential expression between
lumbar and cervical specimens was noted, with significantly increased
expression of LSGAL3, CHRD and NOGGIN noted in cervical NP cells
compared to lumbar NP cells, whilst expression of all other genes
remained unchanged (Figure 3).
DISCUSSION:
This study corroborates other recent evidence regarding the
developmental origins of the NP, as the finding of NC marker
expression in adult NP is indicative of cells of notochordal derivation.
The finding of continuous expression of most markers with ageing and
degeneration may be indicative of an NC-like population of cells that
persist in adult tissues. Interestingly, data shown here suggests that
levels of NC marker expression vary with location within the spine, with
cervical NP cells demonstrating significantly upregulated expression of
multiple NC markers. The reasons for this are unclear, but might suggest
that the persistence of NC-like cells is prolonged in cervical discs and
NC cells of that region might be less susceptible to apoptosis as
previously described. Further work may establish alterations in NC
marker expression across a greater range of ages to include younger
samples where NC cells are known to reside within the NP.
Relative Gene Expression
(Log Scale)
Non-Degenerate
Mildly Degenerate
Moderately Degenerate
10
*
1
*
0.1
0.01
0.001
0.0001
0.00001
T
LSGAL3
CD24
CHRD
FOXA2 NOGGIN
Figure 2 NC Marker Expression According to Age in the Lumbar
NP
100
Young Adult
Relative Gene Expression
(Log Scale)
The anatomical origin of the nucleus pulposus (NP) has been subject
of much debate, with one school of thought suggesting that NP cells
derive from cells of the surrounding AF which is mesenchymal in origin,
whilst other often compelling evidence suggests the NP cells are
notochordally-derived. Morphologically identifiable notochordal cells
have been reported to disappear from the NP after the age of 10 years.
However, as morphological changes in such cells are known to occur,
other methods need to be employed to identify whether notochordallyderived cells exist within the adult NP. The aim of this study was to
investigate the gene expression of previously described notochordal cell
(NC) markers in the adult NP.
Figure 1 NC Marker Expression According to Degeneration in the
Cervical NP
100
Mature Adult
Elderly Adult
10
1
0.1
0.01
0.001
0.0001
1E-05
T
LSGAL3
CD24
CHRD
FOXA2 NOGGIN
Figure 3 Expression of NC Markers in the Lumbar and Cervical NP
100
Relative Gene Expression
(Log Scale)
INTRODUCTION:
Low back pain (LBP) affects 60-80% of individuals in developed
societies at some point, and thus is a major cause of morbidity.
Degeneration of the intervertebral disc (IVD) has been implicated in the
pathophysiology of LBP, but current therapeutic interventions are
largely conservative, involving pain management and mobility-reducing
surgery. Novel cell-based regenerative therapies are being developed,
but in order for these to be successful it is necessary to fully understand
the developmental origins of the NP so that the tissue is restored to that
of a healthy structure and correct functionality.
Cervical Moderately Degenerate
10
Lumbar Moderately Degenerate
*
1
*
*
0.1
0.01
0.001
0.0001
1E-05
T
LSGAL3
CD24
CHRD
FOXA2
NOGGIN
SIGNIFICANCE:
The findings presented here support other evidence suggesting that
the NP derives from the notochord and not the surrounding AF. The
detection of NC markers in adult human NP samples may also indicate
the persistence of a sub-population of cells associated with development
in adult tissues.
ACKNOWLEDGEMENTS:
The authors wish to thank DePuy Spine Inc. for funding this research.
REFERENCES:
1
Sive, J; Baird, P; Jeziorsk, M; Watkins, A; Hoyland, JA and
Freemont AJ. Journal of Clinical Pathology: Molecular Pathology
2002; 55: 91-97
Poster No. 1196 • ORS 2012 Annual Meeting